Tubulysins are members of a new class of natural products isolated from myxobacterial species (F. Sasse, et al., J. Antibiot. 2000, 53, 879-885). As cytoskeleton interacting agents, the tubulysins are mitotic poisons that inhibit tubulin polymerization and lead to cell cycle arrest and apoptosis (H. Steinmetz, et al., Chem. Int. Ed. 2004, 43, 4888-4892; M. Khalil, et al., ChemBioChem. 2006, 7, 678-683; G. Kaur, et al., Biochem. J. 2006, 396, 235-242). Tubulysins are extremely potent cytotoxic molecules, exceeding the cell growth inhibition of any clinically relevant traditional chemotherapeutic, including epothilones, paclitaxel, and vinblastine. Furthermore, tubulysins are potent against multidrug resistant cell lines (A. Dömling, et al., Mol. Diversity 2005, 9, 141-147). These compounds show high cytotoxicity tested against a panel of cancer cell lines with IC50 values in the low picomolar range; thus, they are of interest as potential anticancer therapeutics.
Tubulysins and processes for preparing tubulysins are described herein. In one embodiment, tubulysins and processes for preparing them are described herein that include linear tetrapeptoid backbones, including illustrative compounds having formula T or TE
and pharmaceutically acceptable salts thereof; wherein
Ar1 is optionally substituted aryl or optionally substituted heteroaryl;
R1 is hydrogen, optionally substituted alkyl, optionally substituted arylalkyl or a pro-drug forming group;
R2 is selected from the group consisting of optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted arylalkyl, and optionally substituted heteroarylalkyl;
R12 is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, arylalkyl or heteroarylalkyl, each of which is optionally substituted;
R3 is optionally substituted alkyl;
R4 is optionally substituted alkyl or optionally substituted cycloalkyl;
R5 and R6 are each independently selected from the group consisting of optionally substituted alkyl and optionally substituted cycloalkyl;
R7 is hydrogen or optionally substituted alkyl; and
n is 1, 2, 3, or 4.
In another embodiment, tubulysin amide and hydrazide derivatives, and processes for preparing them are described herein, such as compounds of the following formulae
and pharmaceutically acceptable salts thereof; wherein R1, R2, R12, R3, R4, R5, R6, R7, and n are each independently selected from any of the various embodiments described herein; and m is 1 or 2.
In another embodiment, tubulysins and processes for preparing them are described herein that comprise one or more radicals formed from N-methyl pipecolic acid (Mep), isoleucine (Ile), and/or one or more non-naturally occurring or hydrophobic amino acid segments, such as
and analogs and derivatives of each of the foregoing. Without being bound by theory, it is believed herein that a common feature in the molecular architecture of the more potent natural occurring tubulysins is the acid and/or base sensitive N-acyloxymethyl substituent (or a N,O-acetal of formaldehyde) represented by R2—C(O) in the formula (T).
In another embodiment, tubulysins and processes for preparing them are described herein that have formula 1, and are naturally occurring.
                Formula 1, Structures of several natural tubulysins        
TubulysinRAR2AOHCH2CH(CH3)2BOHCH2CH2CH3COHCH2CH3DHCH2CH(CH3)2EHCH2CH2CH3FHCH2CH3GOHCH═C(CH3)2HHCH3IOHCH3
A total synthesis of tubulysin D has been reported (see, Peltier et al., J Am Chem Soc 128:16018-19 (2006)). Recently, a modified synthetic protocol toward the synthesis of tubulysin B has been reported (Pando et at., Org Lett 11:5567-69 (2009)). However, attempts to follow the published procedures to provide larger quantities of tubulysins were unsuccessful, and were hampered by low yields, difficult to remove impurities, the need for expensive chromatographic steps, and/or the lack of reproducibility of several steps, among other things. The interest in using tubulysins for anticancer therapeutics accents the need for reliable and efficient processes for preparing tubulysins, and analogs and derivatives thereof. Described herein are improved processes for making tubulysins, or analogs or derivatives thereof, including compounds of formula T and TE.
In one illustrative embodiment of the invention, processes for preparing tubulysins, or analogs or derivatives thereof, including compounds of formula T and TE, are described. The processes include one or more steps in the following embodiments.
In another embodiment, a process is described for preparing a compound of formula B, wherein R5 and R6 are independently selected from any of the various embodiments described herein, and R8 is C1-C6 n-alkyl or arylalkyl, such as benzyl. The process comprises the step of treating a compound of formula A with a silylating agent, such as triethylsilyl chloride, and a base, such as imidazole in an aprotic solvent.

In another embodiment, a process is described for preparing a compound of formula C, wherein R2, R5, R6, and R8 are each independently selected from any of the various embodiments described herein. The process comprises the step of treating a compound of formula B with a base and a compound of the formula ClCH2OC(O)R2 in an aprotic solvent at a temperature below ambient temperature, such as in the range from about −78° C. to about 0° C.; wherein the molar ratio of the compound of the formula ClCH2OC(O)R2 to the compound of formula B from about 1 to about 1.5.

In another embodiment, a process is described for preparing a compound of formula D, wherein n, R2, R5, R6, and R8 are each independently selected from any of the various embodiments described herein, and R7 is optionally substituted alkyl. The process comprises the steps of
a) preparing a compound of formula (E1) where X1 is a leaving group or acyl activating group from a compound of formula E; and
b) treating a compound of formula C under reducing conditions in the presence of the compound of formula E1.

In another embodiment, a process is described for preparing a compound of formula FE, wherein n, R2, R5, R6, R7, and R8 are each independently selected from any of the various embodiments described herein. The process comprises the step of contacting compound D with an alcohol, R12OH, where R12 is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which is optionally substituted; and a transesterification catalyst. In one embodiment the transesterification catalyst is trifluoroacetic acid (TFA). In another embodiment, the transesterification catalyst is selected from the group consisting of (R13)8Sn4O2(NCS)4, (R13)2Sn(OAc)2, (R13)2SnO, (R13)2SnCl2, (R13)2SnS, (R13)3SnOH, and (R13)3SnOSn(R13)3, where R13 is independently selected from alkyl, arylalkyl, aryl, or cycloalkyl, each of which is optionally substituted. In another embodiment, the transesterification catalyst is (R13)2SnO. Illustrative examples of R13 are methyl, n-butyl, n-octyl, phenyl, o-MeO-phenyl, p-MeO-phenyl, phenethyl, benzyl, and the like.

In another embodiment, a process is described for preparing a compound of formula IE, wherein n, R5, R6, R7, and R12 are each independently selected from any of the various embodiments described herein. The process comprises the step of contacting compound FE with a metal hydroxide or carbonate. Illustrative examples of a metal hydroxide or carbonate include LiOH, Li2CO3, NaOH, Na2CO3, KOH, K2CO3, Ca(OH)2, CaCO3, Mg(OH)2, MgCO3, and the like.

In another embodiment, a process is described for preparing a compound of formula G, wherein n, R2, R5, R6, R7, and R8 are each independently selected from any of the various embodiments described herein. The process comprises the step of treating compound D with a hydrolase enzyme.

In another embodiment, a process is described for preparing a compound of formula I, wherein n, R2, R5, R6, and R7 are each independently selected from any of the various embodiments described herein. The process comprises the step of treating the silyl ether of compound G with a non-basic fluoride containing reagent.

In another embodiment, a process is described for preparing a compound of formula GE, wherein n, R2, R5, R6, and R7 are each independently selected from any of the various embodiments described herein. The process comprises the step of contacting compound G with an alcohol, R12OH, and a transesterification catalyst, as described herein.

In another embodiment, a process is described for preparing a compound of formula IE, wherein n, R5, R6, R7, and R12 are each independently selected from any of the various embodiments described herein. The process comprises the step of treating the silyl ether of compound GE with a non-basic fluoride containing reagent.

In another embodiment, a process is described for preparing a compound of formula CE, wherein R5, R12, R6, and R8 are each independently selected from any of the various embodiments described herein. The process comprises the step of treating a compound of formula B with a base and a compound of the formula YCH2OR12, where Y is a leaving group such as a halogen selected from chloro, bromo, and iodo, in an aprotic solvent at a temperature below ambient temperature, such as in the range from about −78° C. to about 0° C.; wherein the molar ratio of the compound of the formula YCH2OR12 to the compound of formula B from about 1 to about 1.3.

In another embodiment, a process is described for preparing a compound of formula DE, wherein n, R12, R5, R6, and R8 are each independently selected from any of the various embodiments described herein, and R7 is optionally substituted alkyl. The process comprises the steps of
a) preparing a compound of formula (E1) where X1 is a leaving group or acyl activating group from a compound of formula E; and
b) treating a compound of formula CE under reducing conditions in the presence of the compound of formula E1.

In another embodiment, a process is described for preparing a compound of formula IE, wherein n, R12, R5, R6, R7, and R8 are each independently selected from any of the various embodiments described herein. The process comprises the step of treating the silyl ether/ester of compound DE with a base.

In another embodiment, a process is described for preparing a compound of formula JE, wherein n, R12, R4, R5, R6, and R7 are each independently selected from any of the various embodiments described herein. The process comprises the step of treating a compound of formula IE with an acylating agent of formula R4C(O)X2, where X2 is a leaving group.

In another embodiment, a process is described for preparing a tubulysin of formula (TE), wherein n, Ar1, R1, R3, R4, R5, R6, R7, and R12 are each independently selected from any of the various embodiments described herein. The process comprises the step of forming an active ester intermediate from a compound of formula JE; and reacting the active ester intermediate with a compound of the formula M to give a compound of the formula TE.

In another embodiment, a process is described for preparing a tubulysin linker derivative of formula (TE-L-X), wherein n, Ar1, R1, R3, R4, R5, R6, R7, and R12 are each independently selected from any of the various embodiments described herein;
L is selected from the group consisting of
where p is an integer from about 1 to about 3, m is an integer from about 1 to about 4, and * indicates the points of attachment; where
Ra, Rb, and R are each independently selected in each instance from the group consisting of hydrogen and alkyl; or at least two of Ra, Rb, or R are taken together with the attached carbon atoms to form a carbocyclic ring;
RAr represents 0 to 4 substituents selected from the group consisting of amino, or derivatives thereof, hydroxy or derivatives thereof, halo, thio or derivatives thereof, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, nitro, sulfonic acids and derivatives thereof, carboxylic acids and derivatives thereof; and
X is an activated sulfur that includes a leaving group that allows formation of a bond between the sulfur and a nucleophile.
The process comprises the step of contacting compound T-L-X, with an alcohol, R12OH, and a transesterification catalyst, as defined herein.
Compounds of formula T-L-X are prepared according to the processes described in PCT international application serial No. PCT/US2013/034672, the disclosure of which is incorporated herein by reference. In another embodiment, the transesterification catalyst is TFA. In another embodiment, X is SS—Ar2, where Ar2 is optionally substituted aryl or optionally substituted heteroaryl. In another embodiment, Ar2 is selected from optionally substituted pyridin-2-yl, pyridin-2-yl, nitrophenyl, and the like.
In another embodiment, a process is described for preparing a tubulysin linker derivative of formula (TE-L-X), wherein L, X, n, Ar1, R1, R2, R3, R4, R5, R6, R7, and R12 are each independently selected from any of the various embodiments described herein. The process comprises the step of forming an active ester intermediate KE, where Y is a leaving group, from a compound of formula JE; and reacting the active ester intermediate KE with a compound of the formula M-L-X to give a compound of the formula TE-L-X.
Compounds of formula M-L-X are prepared according to the processes described in PCT international application serial No. PCT/US2013/034672, the disclosure of which is incorporated herein by reference.
It is to be understood that in each process or process step described hereinabove or hereinbelow, each group L, X, n, Ar1, R1, R2, R3, R4, R5, R6, R7, and R12 is independently selected in each instance from any of the various embodiments, genera, subgenera, or lists described herein. For example, in another illustrative embodiment of any of the processes or process steps described hereinabove or hereinbelow, Ar1 is optionally substituted aryl. In another illustrative embodiment of any of the processes or process steps described hereinabove or hereinbelow, Ar1 is optionally substituted heteroaryl.
It is also to be understood that each of Ar1, Ar2, R1, R2, R12, R3, R4, R5, R6, and R7 may include any necessary or desirable conventional protection groups as appropriate for the reaction conditions in the corresponding process step.